Fluorescent fatty acids and derivatives of fluorescent fatty acids are examples of what are usually termed fluorescent probes. A fatty acid is a hydrocarbon chain terminating in a carboxylic acid. The hydrocarbon chain may be completely saturated or may contain one or more double bonds; the chain may be linear or branched. A fluorescent probe is a fluorescent compound used to "probe" structures and/or processes at the molecular and cellular level, by mimicking the natural compound. Fluorescent probes have been extensively utilized in biological and biomedical research both to visualize sites and for quantitative measurements. Fluorescent probes have also been used in electronics, polymer chemistry, medicine, forensics, and other diverse areas.
Fluorescent fatty acids have been used as probes for membrane and liposome structure and biological properties, for biosynthetic incorporation into cells, and in Langmuir-Blodgett films, etc. Fluorescent fatty acids have been synthetically incorporated into triglycerides, phospholipids, cholesteryl esters, phorbols, sphingolipids, and coenzyme A. Such synthetic products from fluorescent fatty acids have applications as probes similar to the fluorescent free fatty acids. The synthetic product to which the fluorescent fatty acid is attached, however, may also confer ability to specifically interact with a receptor site. This site may be a binding site on a cell, an enzyme, a polymer or other structure. The presence of the fluorophore as part of the probe may permit detection or quantitation of binding to the receptor, following the kinetics of an enzyme-catalyzed reaction, measurement of properties of the site to which the probe is bound, or visualization of substrates to which the probe is bound.
Fluorescence useful for such applications is generally initiated by absorption of light from an external, relatively concentrated light source. The sensitivity of these applications is improved by having dyes that have high absorbance of the exciting light and high fluorescence quantum yield. The applications are furthermore improved by having dyes that resist photobleaching by the exciting light and that have spectral wavelengths in a range that avoids the background from contaminants that may be present in the samples.
Certain lasers are particularly useful as a concentrated light source for the excitation of fluorescence. These include the argon laser with principal output at 488 nm and 514 nm; helium-neon lasers that can be selected to have maximum output at either 543 nm, 594 nm, or 633 nm; the krypton laser which has significant output at 568 nm and 647 nm; and laser diodes, which are commonly available at this time, with output above 660 nm. The argon laser is extensively used in such techniques as flow cytometry and laser scanning microscopy, which are two areas in which this invention has applications.
A number of dyes have previously been found to be fluorescent, however many of these dyes have characteristics which interfere with their usefulness. For example, many known fluorescent dyes do not have significant absorbance at the desired excitation wavelengths, or are significantly quenched in aqueous solution or are unstable during the illumination.
Fluorescent analogs of fatty acids have been described previously. Among those described are those derived from the fluorescent compounds anthracene (Waggoner and Stryer, Fluorescent Probes of Biological Membranes, PROC. NATL. ACAD. SCI. 67, 579 (1970) and Bony, Lopez, Gilleron, Welby, et.al., Transverse and Lateral Distribution of Phospholipids and Glycolipids in the Membrane of the Bacterium Micrococcus luteus, BIOCHEM. 28, 3728 (1989)), pyrene (Galla & Sackmann, Chemically Induced Phase Separations in Mixed Vesicles Containing Phosphatidic Acid. An Optical Study, J. AM. CHEM. SOC. 97, 4114 (1975)), perylene (Shinitzky, Dianoux, Gitler & Weber, Microviscosity and order in the mydrocarbon region of micelles and membranes determined with fluorescent probes. I. Synthetic micelles, BIOCHEM. 10, 2106 (1971)), naphthalene (Lee et.al., Interaction of Fatty Acids with the Calcium-Magnesium Dependent Adenosine Triphosphatase from Sarcoplasmic Reticulum, BIOCHEM. 21, 6441 (1982)), carbazole (Omann & Lakowicz, Interactions of Chlorinated Hydrocarbon Insecticides with Membranes, BBA 684, 83 (1982)), and nitrobenzoxadiazole (NBD) (Derzko & Jacobson, Comparative Lateral Diffusion of Fluorescent Lipid Analogues in Phospholipid Multibilayers, BIOCHEM. 19, 6050 (1980)).
Of these fluorescent fatty acids that have been described previously, only the NBD analogs have absorption maxima above 450 nm. The NBD derivatives have maximum absorption at 460 to 480 nm. Long wavelength absorbance usually increases the utility of a fluorescent probe since it reduces the interference from cellular autofluorescence and reduces the cytotoxic effect of the fluorophore in combination with light. Furthermore, absorbance at 488 or 514 nm permits use of the principal output of the argon laser to effect excitation of the probe.
Known fluorescent fatty acids have other disadvantages as well. Fluorescence of NBD derivatives, for example, is highly dependent on the environment. The NBD fluorophore also tends to fold back to the aqueous interface in cell membranes and liposomes. See Chattopadhyay & London, Spectroscopic and Ionization Properties of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-Labeled Lipids in Model Membranes, BIOCHIM. BIOPHYS. ACTA 938, 24 (1988). Other long-wavelength fluorophores such as fluoresceins, rhodamines, carbocyanines, oxonols, and most oxazines have electronic charges at pH 7 which usually preclude their use as lipophilic fluorescent probes because the fluorophore is not readily incorporated into the lipophilic environment of the membrane.
What is needed is an improved fatty acid analog. Dipyrrometheneboron difluoride ("BDY") dyes contribute many desirable characteristics to the fatty acid analogs. Longer wavelength fatty acids derived from BDY, which are the subject of this invention, extend the useful excitation wavelengths from near 500 nm to beyond 600 nm. The BDY fluorophore is electrically neutral and lipophilic, which properties permit it to be solubilized better in non-polar solvents and cell membranes. The BDY fluorophore also has significantly higher absorbance and fluorescence yield than any of the other fluorophores that have been used to prepare fluorescent fatty acids. The BDY fatty acids are also highly colored and may be detected without utilizing the fluorescent characteristic.
Simple alkyl derivatives of the BDY fluorophore, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, have been described by Treibs & Kreuzer, Difluorboryl-komplexe von diund tripyrrylmethenenLIEBIGS ANNALEN CHEM. 718, 203 (1968) and by Worries, Kopek, Lodder, & Lugtenburg, A novel water-soluble fluorescent probe: Synthesis, luminescence and biological properties of the sodium salt of the 4-sulfonato-3,3',5,5'- tetramethyl-2,2'--pyrromethen-1,1'-BF.sub.2 complex, RECL. TRAV. CHIM. PAYS-BAS 104, 288 (1985) as being highly fluorescent with spectral properties that are similar to fluorescein with maximum absorbance at about 490 to 510 nm and maximum emission at about 500 to 530 nm. U.S. Pat. No. 4,774,339 to Haugland et al. (1988) ('339 patent) describes BDY dyes that contain reactive groups suitable for conjugation to biomolecules, that have good photostability, and which have fluorescein-like spectra. Heteroaryl-Substituted Dipyrrometheneboron Difluoride Dyes and Methods for Their Synthesis are described in co-pending application filed by inventors Haugland and Kang on Dec. 18, 1990 ("heteroaryl application"). Neither the earlier references nor the '339 patent nor the heteroaryl application disclose nor suggest the subject BDY fatty acids with long wavelength fluorescence properties, nor the use of BDY dyes to form fatty acid analogs.
It is the object of this invention to provide novel fluorescent fatty acid analogs useful as probes, and methods for their synthesis. It is a further object of this invention to provide novel fluorescent fatty acids derived from the BDY fluorophore.